Available methods for molecular structure determination, based primarily on x-ray crystallography and Nuclear Magnetic Resonance (NMR) solution methods, have had limited success on the insoluble proteins that are critical to biological function. Various recent developments have enhanced the effectiveness of solids NMR methods incorporating Magic Angle sample Spinning (MAS), and considerable additional progress in such techniques continues. Yet, the fact remains that stationary (non-MAS) high-power methods, such as PISEMA, have been more fruitful thus far in yielding structures of large, complex, helical membrane proteins. Preliminary work recently published by several leading research groups has demonstrated the value of advanced 3D methods that cannot be carried out using any commercially available probes, and can only be marginally implemented on home-built probes in mid-field magnets in a few laboratories. Several of the world's most prestigious and successful researchers in macromolecule structure determination by solids NMR methods have voiced the need for major increases in RF field strength, as required for significantly improved spectral resolution, along with dramatically reduced RF sample heating, in triple-resonance 1H/13C/15N probes. This Phase II proposal seeks funding to complete the development of an ultra-high-power triple-resonance probe for fields up to 1 GHz with order-of-magnitude reduction in RF sample heating and more than a factor of two improvement in each of the remaining three most important and technically demanding specifications simultaneously: RF field strength, spectral resolution, and S/N. The net result is expected to be an order of magnitude reduction in signal acquisition time for many techniques in biological macromolecules. The Phase I effort demonstrated the feasibility of the approach based on a prototype 5-mm probe for 500 MHz and simulations at 900 MHz. The Phase II, 4 mm, 900 MHz probe is expected to demonstrate the following: (1) ability to generate sustained rotating-frame frequencies above 110 kHz at the three resonances simultaneously, (2) static spectral resolution below 0.02 ppm, and (3) S/N on 15N better than 50:1 on 70 <L of natural- abundance formamide. Achieving the desired RF field strengths will require 4 kW RF pulses for 15N (91 MHz), 1500 W RF pulses for 13C (226 MHz), and 350 W RF pulses for 1H (900 MHz). The approach will be compatible with operation in narrow-bore (NB) magnets at the highest fields anticipated in the coming decade - to 1.0 GHz. The proposed work builds on earlier work in reducing RF sample heating and improving power handling and resolution in MAS probes;and it adds proprietary, novel technologies to achieve record-shattering power handling. Initial field testing of the Phase II 900 MHz triple-resonance PISEMA probe is expected before the end of the first year at the National High Magnetic Field Laboratory in Florida. PUBLIC HEALTH RELEVANCE: There is strong medical and scientific interest in determining the structures of the 15,000 membrane proteins in the human body over the next decade, though available NMR and X-ray methods work poorly and have yielded only a few such structures over the past decade. There are more than 5,000 high-field NMR systems installed world-wide, and annual NMR equipment sales are currently ~$300M. The proposed ultra-high-power NMR probe development is expected to enhance the ability to determine molecular structures of large, insoluble, membrane proteins by advanced NMR methods by an order of magnitude in many cases.